All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Biomolecules in general and proteins in particular are capable of self-assembling into a wide variety of structures that can be readily manipulated and functionalized. Today there is an exceeding demand in the field of nano-biotechnology and material science for a reliable, highly stable building block that can self assemble to form higher hierarchy ordered compounds.
Biopolymer research has focused in recent years on fibrous proteins due to their unique mechanical properties. For example, one can mention the mammalian collagen and elastin and the arthropod proteins, silk worm silk (Bombyx morii), spider dragline silk and resilin. These proteins are distinguished by their repetitive amino acid sequences that confer their mechanical properties such as strength and flexibility. For instance spider silk is extremely strong while resilin and elastin are extremely stretchy/elastic and resilient and have a rubber like nature. The unique repetitive sequence of each protein confers its three-dimensional architecture and mechanical properties. Much can be learned from the organizational principles employed in native biopolymers. Substantial progress has been made in the elucidation of the three-dimensional architectures of spider silks and collagens. Still, an important challenge is the translation of these concepts into synthetic or bio-inspired materials, which would lead to new kinds of high performance fibers. The translation process requires a level of control of macromolecular architecture far higher than that afforded by conventional polymerization processes. These biomaterials could replace, in part, synthetic plastics, fibers and elastomers, thus offering the advantage of renewability, sustainability and biodegradability.
Cellulose is degraded in nature by the concerted action of a number of bacterial and fungal organisms. The initial event in the degradation process is the binding of the cellulolytic enzyme(s) or the entire microorganism to the cellulose substrate. A separate domain, named CBD, mediates this binding. The CBD enables adhesion of the water soluble enzyme onto an insoluble substrate surface (cellulose). The close association between the enzyme and cellulose provided by the CBD enhances the catalytic rate and stability of the enzyme. A wide variety of CBDs are known, having different binding constants as well as sensitivity to heat and pH. The binding domains are classified into 61 different families (see http://www.cazy.org), based on amino acid sequences, binding specificity, and structure. The binding of Clostridium cellulovorans CBD (CBDclos, family III) is unique in the manner in which it maintains its specific cellulose binding properties under conditions which most proteins are denatured and nonfunctional. The CBDcex from Cellulomonas fimi (family II) binds to many forms of insoluble cellulose, including amorphous, semi-crystalline, and crystalline and was applied in a variety of fusion proteins.
It is well known that some kinds of CBDs form dimers or higher molecular weight compounds in solution or upon binding to cellulose. Different proteins fused to CBDclos were expressed in E. coli and were shown to form dimers in solution [Xu Y. et al., J. Biotechnol. 135: 319-25 (2008)]. Chromatography, ultracentrifugation and 15N NMR relaxation experiments demonstrate that CBDcex is a dimer in solution [Xu G. Y. et al., Biochemistry 34:6993-7009 (1995)].
Silk proteins are produced by a variety of insects and arachnids, the latter of which form the strongest silk polymers on earth. The spider spins as many as seven different kinds of silks, each one being optimized to its specific biological function in nature. Dragline silk, used as the safety line and as the frame thread of the spider's web, is an impressive material with a combination of tensile strength and elasticity. These properties endow it with unique toughness which displays the highest energy to break among common natural or artificial materials. The dragline fiber is predominantly proteineacous, comprised of greater than 80% glycine, alanine and other short side chain amino acids with crystalline regions of antiparallel β-pleated sheets. The dragline fiber's extraordinary properties are derived from its composition as a semicrystalline polymer, comprising crystalline regions embedded in a less organized “amorphous” matrix. The antiparallel β-pleated sheets of polyalanine stretches give strength to the thread, while the predominant secondary structure of the amorphous matrix is the glycine-rich helix which provides elasticity. Most dragline silks consist of at least two different proteins with molecular masses of up to several hundred kDa.
Numerous gene sequences coding for spider type silks are known [Gatesy et al., Science 291:2603-2605 (2001)]. An examination of the cDNAs, genes and amino acid sequences shows that all silks are chains of iterated peptide motifs. The consensus sequences for the repeating peptides are repeated multiple times throughout the core of each protein, flanked by non repetitive, highly conserved, short terminal sequences. On the basis of sequence similarities, dragline silk proteins have been grouped into spidroin1-like (MaSp1) and spidroin2-like (MaSp2) proteins.
As opposed to silkworm silk, isolation of silk from spiders is not industrially feasible. Spiders produce silk in small quantities, and their territorial behavior prevents large amounts thereof from being harvested in adjacent quarters. Therefore, production of silk protein through recombinant DNA techniques is preferred. For such purposes, widespread use is made of synthetic genes based on a monomer consensus of the native spidroin sequences. These synthetic genes have been successfully expressed in the methyltropic yeast host, Pichia pastoris, in E. coli and in the tobacco and potato plants [Fahnestock et al., Appl. Microbiol. Biotechnol. 47:33-39 (1997); Fahnestock et al., Appl. Microbiol. Biotechnol. 47:23-32 (1997); Sceller et al., Nature biotechnology 19:573-577 (2001)]. Through such means, laboratory scale amounts of silk-like protein powders are readily available. The final hurdle on the way to the production of manmade silks lies in the development of an appropriate spinning technology capable of converting these powders into high performance fibers. A significant limitation toward successfully producing functional silk has been the tendency of these proteins to aggregate in-vitro, bypassing the protein folding process. The assembly of the proteins in a liquid crystalline form into a solid silk string is extremely complex, and duplication of the operational function of spider spinning glands is a major challenge. The present inventors have previously shown generation of composites comprising fusion of fibrous proteins to CBD attached to cellulose [US 2010/0317588].
The present invention now discloses a method utilizing CBD's ability to form dimers or higher molecular weight compounds in order to direct molecular order and assembly of CBD fusion proteins. The invention specifically demonstrates CBD's ability to create higher hierarchy ordering by its fusion to dragline spider silk proteins.
Thus, one object of the invention is to provide a method for generating high molecular ordered fibrilar structures by directing ordered assembly of fibrous proteins.
Another object of the invention is to provide isolated high molecular ordered fibrilar structures.
Still further the invention provides the use of the fibrilar structures in medicine, in reconstruction of tissues, as scaffolds for growth of cells, as support for bone, ligaments and tendon, and/or as parts of implantable medical device. The invention further provides the use of these fibrilar structures in the military and avian industry where high strength and low weight are important, e.g. in unmanned aerial vehicle, in personal armor and the like.
These and other objects of the invention will become apparent as the description proceeds.